In the production of proteins and polypeptides, either by extraction or by recombinant biotechnology techniques, there is the problem of maintaining the correct folding of the protein so as to keep its desired activity.
Unfolding or incorrect or otherwise modified foldings may occur because of technical manipulation or the general processing system for the production of the proteins.
Another problem in proteinaceus material processing is given by the aggregation of proteins.
Many solutions are offered in the state of the art. Some of them are peculiarly chemical, meaning by this that chemical reagents are used, such as, for example particular mixtures of salts, even in buffer solutions.
U.S. Pat. No. 5,728,804, to research Corporation Technologies, discloses a method for protein renaturation by means of detergent-free cyclodextrins. U.S. Pat. No. 5,563,057, to Wisconsin Alumni Research Foundation, other than cyclodextrin, teaches the use of certain detergents for refolding misfolded enzymes.
U.S. Pat. No. 5,874,075, to Amgen, discloses protein:phospholipids complexes useful for stabilizing proteins against thermally-induced aggregation, denaturation and loss of activity.
U.S. Pat. No. 5,756,672, to Genentech, provides a composition comprising a polypeptide in a certain buffer. Said buffer being suitable for refolding improperly folded polypeptides. A particular embodiment is given for refolding misfolded insulin-like growth factor-I.
The above mentioned methods might be convenient, since easily available chemicals are used, but may raise some instances for certain chemicals used, for example copper or manganese salts (U.S. Pat. No. 5,756,672).
Refolding occurs also through chromatographic techniques, See Altamirano M M. Et al. Nat. Biotechnol. 1999 February; 17(2):187-91.
The discovery of chaperonins has opened a new field for the technology of protein processing.
Chaperonins, also known as heat-shock proteins or HSP; are natural proteins exerting a biological role in protein folding. See for an extensive review internet address ermm.cbcu.cam.ac.uk/000021015h.htm by Julia C. Ranford, Anthony R. M. Coates and Brian Handerson.
Technically, chaperonins are intensively studied as means for facing the above-mentioned problem of protein stabilization and refolding.
This search leads to newly discovered chaperonins and to their use for protein stabilization, see for example U.S. Pat. No. 5,428,131, to Yale University. For a picture of chaperonins for the technical problem faced by the present invention, see for example U.S. Pat. No. 5,688,651, to RAMOT University; U.S. Pat. No. 5,646,249, to U.S. Health Department; U.S. Pat. No. 5,561,221, to Nippon Oil Company Limited, WO 00/20606, to Reiman and Schirmbeck, J P 11266865, to Kaiyo Biotechnology Kenkyusho K K, WO 99/40435, to Netzer; JP 10327869, to Kaiyo Biotechnology Kenkyusho K K; WO 00/71723, to Roche Diagnostics; WO 00/55183 and WO 99/05163, to Medical Research Council.
Chaperonins are a useful tool for protein stabilization and refolding, but some technical drawbacks come from their use. Since they are proteins, even they are prone to alteration, such as thermal one, so they too need some protective factor.
For the technical field of stabilizing proteins, this problem is also very important for preparation of HSP cancer vaccines. It has been observed that the immunogenicity of a given antigen is rendered far more efficient when it is presented to immune cells in a complex with HSPs (Requena J M et al, Ars-Pharm 1997, 38(2-3):191-208; Castellino F, et al, J Exp Med 2000, 191(11):1957-1964). Particularly, the immune response to cancer is boosted with HSP (i.e., HSP70 or gp96) which are linked to an antigenic peptide (“specific antigenic fingerprints”), both of which are obtained from the patient's cancer cells (Yedavelli Spet al Int J Mol Med 1999, 4(3):243-248). It is therefore important to have stable and/or well preserved HSP for cancer vaccines.
Interestingly, some chaperoning, such as the eye lens alpha-crystallin proteins, are members of the small heat shock protein (sHSP) family. sHSPs have been shown to function in a number of different processes ranging from RNA stabilization to elastase inhibition and interaction with the cytoskeleton.
AlphaA-crystallin is localized primarily in the lens with very low levels found in other tissues, whereas alphaB-crystallin is now known to be essentially ubiquitous throughout the body (Haley D A et al, J Mol Biol 1998, 277:27-35). The biological importance of alphaB-crystallin is highlighted by its elevated levels in ischemic heart and in the brains of patients with multiple sclerosis, Alzheimer's and other neurological diseases. Consistent with its classification as an HSP, expression of alphaB-crystallin has been shown to be induced by a variety of physiological stresses including heat, osmotic stress, and metal toxicity. The biological importance of alphaA-crystallin in lens is highlighted by its efficient suppression of uncontrolled aggregation of damaged proteins.
As it appears from the above examples, the stabilization of chaperoning, either for their use or for stabilizing them in those pathological states in which their are altered, is very important in the medical field.